This invention relates to methods for the preparation of oligomeric compounds having pendant groups conjugated thereto through a novel haloacetyl linker. This invention also relates to compounds containing such haloacetyl linkers, and to methods and intermediates useful for their preparation.
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The chemical literature discloses numerous processes for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most popular processes is the phosphoramidite technique (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
Synthetic oligonucleotides having 5xe2x80x2-terminal functional or reporter groups are employed in a number of bioanalytical and antisense applications. Appropriately, a number of procedures and reagents that allow their preparation have been described, including conjugation of electrophiles to oligonucleotides via tethers carrying nucleophilic groups. For the opposite process of conjugating nucleophiles to electrophilic sites, the methods available to generate these sites in oligonucleotides are limited. These include periodate oxidation of a terminal ribose moiety or the use of an internal abasic site within the oligonucleotide sequence. Heterobifunctional reagents that bear a phosphoramidite moiety along with an electrophilic functional group that is reactive in orthogonal conditions are therefore of particular interest.
One group have reported methods that utilize the reactivity of (thio)ester group towards alkylamines. See Hovinen, J., Guzaev, A., Azhayeva, E., Azhayev, A. and Lxc3x6nnberg, H. J. Org. Chem. 1995, 60, 2205; Azhayeva, E., Azhayev, A., Guzaev, A., Hovinen, J. and Lxc3x6nnberg, H. Nucleic Acids Res. 1995, 23, 1170; Guzaev, A., Hovinen, J., Azhayev, A. and Lxc3x6nnberg, H. Nucleoides and Nucleotides 1995, 14, 833; Hovinen, J., Guzaev, A., Azhayev, A. and Lxc3x6nnberg, H. J. Chem. Soc. Perkin Trans. I 1994, 2745; and Hovinen, J., Guzaev, A., Azhayev, A., and Lxc3x6nnberg, H. Tetrahedron, 1994, 50, 7207. On completion of chain elongation, the modified oligonucleotide could be converted into a variety of conjugates by treatment with linkers of different length and substituents that bear a primary amino group.
A haloacetyl linker that is reactive towards a variety of nucleophiles has been introduced previously into oligonucleotides by postsynthetic reaction in solution. See Goodchild, J. Bioconjugate Chem. 1990, 1, 165. Ligands carrying amino groups may be either less available or less desirable for attachment than those with other nucleophilic groups. Therefore, more universal methods for preparation of oligonucleotide conjugates with the aid of heterobifunctional phosphoramidite building blocks are greatly desirable. This invention is directed to this, as well as other, important ends.
In accordance with one aspect of the present invention, methods are provided for the preparation of a conjugated oligonucleotide comprising:
providing a compound of Formula I: 
wherein:
R1 is a phosphorus protecting group;
R2 is chlorine or a pendant group;
R3 is xe2x80x94N(R4)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R4 is straight or branched chain alkyl having from 1 to 10 carbons.
v is 0 or 1;
n is 1 to about 10;
Q has one of the Formulas II or III: 
wherein:
R5 is a hydroxyl protecting group;
B is a nucleobase;
R6 is F, Oxe2x80x94R20, Sxe2x80x94R20 or Nxe2x80x94R20(R21)
R20 is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic;
and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylamino, urea or alkylurea group;
or R2, has one of the formulas: 
wherein
Q1 is O, S or NR2;
m is from 1 to 10;
y is from 0 to 10;
E is N(R21) (R31), Nxe2x95x90C(R21) (R31), C1-C10 alkyl, or C1-C10 substituted alkyl wherein said substituent is N (R2) (R3);
each R21 and R31 is, independently, H, C1-C10 alkyl, alkylthioalkyl, a nitrogen protecting group, or R2 and R3, together, are a nitrogen protecting group or wherein R2 and R3 are joined in a ring structure that can include at least one heteroatom selected from N and O; and R41 is H or C1-C12 alkyl;
reacting said compound of Formula I with a compound of Formula IV: 
wherein:
R7 is H, a hydroxyl protecting group, or a linker connected to a solid support;
w is 0 to about 100;
to form a compound of Formula IVa: 
and
contacting the compound of Formula IVa with a pendant group for a time and under conditions sufficient to form said conjugate.
Some preferred embodiments of the methods of the invention further comprise oxidizing or sulfurizing the compound of Formula IVa to form a compound of Formula IVb: 
wherein R11 is O or S.
In some preferred embodiments, v is 0. In further preferred embodiments, v is 1. In still further preferred embodiments, R2 is Cl. In yet further preferred embodiments, R2 is Cl and R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl.
In further preferred embodiments, R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, and R1 is xcex2-cyanoethyl, xe2x80x94CH2CHxe2x95x90CHCH2CN, para-CH2C6H4CH2CN, xe2x80x94(CH2)2-5N(H)COCF3, xe2x80x94CH2CH2Si(C6H5)2CH3, or CH2CH2N(CH3)COCF3, with xcex2-cyanoethyl being preferred.
In some preferred embodiments, n is 2 to 8, with 4 to 6 being preferred, and 6 being more preferred.
In some preferred embodiments of the methods of the invention, R7 is a linker connected to a solid support.
In some preferred embodiments, the pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
In some preferred embodiments, v is 0, R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, R1 is xcex2-cyanoethyl, n is 6, R7 is a linker connected to a solid support, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
In other preferred embodiments, v is 1, R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, R1 is xcex2-cyanoethyl, n is 6, R7 is a linker connected to a solid support, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid, and Q has the Formula II.
In still further preferred embodiments, V is 1, R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, R1 is xcex2-cyanoethyl, n is 6, R7 is a linker connected to a solid support, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid, and Q has the Formula III.
Also provided in accordnce with the present invention are synthetic methods comprising:
providing a compound of Formula H2Nxe2x80x94(CH2)nxe2x80x94OH wherein n is 1 to about 10;
reacting said compound with a compound of Formula VIII: 
to form a chloroacetylamino alkanol compound of Formula IX: 
and contacting said chloroacetylamino alkanol compound with a reagent of Formula (R3)2Pxe2x80x94Oxe2x80x94R1 
wherein:
R1 is a phosphorus protecting group;
R3 is xe2x80x94N(R4)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R4 is straight or branched chain alkyl having from 1 to 10 carbons.
n is 1 to about 10;
for a time and under conditions sufficient to form an activated phosphate compound of Formula X: 
In some preferred embodiments, R3 is diisopropylamino; and R1 is xcex2-cyanoethyl.
Further preferred embodiments of the methods of the inventikon further comprise reacting said activated phosphate compound with a free hydroxyl group of a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support to form a compound of Formula XI: 
wherein R9 is a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support.
Still further preferred embodiments further comprise oxidizing or sulfurizing said compound of Formula XI to form a compound of Formula XII: 
wherein R11 is O or S.
Still further preferred embodiments further comprise coupling a pendant group to the compound of Formula XII, preferably wherein said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
The invention also provides novel compounds having the Formula V: 
wherein:
R2 is halogen or a pendant group;
R10 is a nucleobase, a nucleoside, a nucleotide, an activated nucleotide, an oligonucleotide, an oligonucleotide connected to a solid support, or a moiety of Formula VI: 
wherein:
R1 is H or a phosphorus protecting group;
R3 is xe2x80x94N(R4)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R4 is straight or branched chain alkyl having from 1 to 10 carbons; and
n is from 1 to about 10.
In some preferred embodiments, R10 is a nucleoside, a nucleotide, an activated nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support. In further preferred embodiments R10 has the Formula IV.
In some preferred embodiments, compounds of the invention have the Formula VII: 
wherein:
v is 0 or 1;
q is 0 or 1;
Q has one of the Formulas II or III: 
wherein:
R5 is a hydroxyl protecting group;
B is a nucleobase;
R6 is F, Oxe2x80x94R20, Sxe2x80x94R20 or Nxe2x80x94R20 (R21)
R20 is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic;
and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylamino, urea or alkylurea group;
or R20 has one of the formulas: 
wherein
Q1 is O, S or NR2;
m is from 1 to 10;
y is from 0 to 10;
E is N(R21)(R31), Nxe2x95x90C(R21)(R31), C1-C10 alkyl, or C1-C10 substituted alkyl wherein said substituent is N(R2)(R3);
each R21 and R31 is, independently, H, C1-C10 alkyl, alkylthioalkyl, a nitrogen protecting group, or R2 and R3, together, are a nitrogen protecting group or wherein R2 and R3 are joined in a ring structure that can include at least one heteroatom selected from N and O; and
R41 is H or C1-C12 alkyl.
In some preferred embodiments, v is 0 and q is 1. In further preferred embodiments, v is 1. In further preferred embodiments, v is 1 and q is 1.
In some preferred embodiments, R2 is Cl. In further preferred embodiments, R2 is Cl and R3 is xe2x80x94N(R4)21 wherein R4 is isopropyl. In still further preferred embodiments, R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, and R1 is xcex2-cyanoethyl, xe2x80x94CH2CHxe2x95x90CHCH2CN, para-CH2C6H4CH2CN, xe2x80x94(CH2)2-5N(H)COCF3, xe2x80x94CH2CH2Si(C6H5)2CH3, or CH2CH2N(CH3)COCF3, with xcex2-cyanoethyl being preferred.
Preferably, n is 2 to 8, with 4 to 6 being more preferred, and 6 being especially preferred.
In some preferred embodiments, v and q are each 1;
and Q has the Formula III wherein the moiety xe2x80x94Oxe2x80x94P(R3)xe2x80x94Oxe2x80x94R1 is attached to B at the N2 position.
In further preferred embodiments, v and q are each 1; and Q has the Formula II wherein the moiety xe2x80x94Oxe2x80x94P(R3)xe2x80x94Oxe2x80x94R1 is attached at the 2xe2x80x2-position.
In some preferred embodiments, said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
In some especially preferred embodiments, v is 0;
q is 1; R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, R1 is xcex2-cyanoethyl, n is 6, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
In further especially prefered embodiments, v and q are each 1; R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, R1 is xcex2-cyanoethyl, n is 6, said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid, and Q has the Formula II wherein the moiety xe2x80x94Qxe2x80x94P(R3)xe2x80x94Oxe2x80x94R1 is attached at the 2xe2x80x2-position.
In yet further especially preferred embodiments R2 is Cl, R3 is xe2x80x94N(R4)2, wherein R4 is isopropyl, R1 is xcex2-cyanoethyl, n is 6, said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid, and Q has the Formula III wherein the moiety xe2x80x94Oxe2x80x94P(R3)xe2x80x94Oxe2x80x94R1 is attached at the N2-position.
In one aspect, the present invention provides methods for the preparation of oligomeric compounds having at least one pendant group conjugated thereto. In accordance with the methods of the invention, conjugation of the pendant group to the oligomeric compound is achieved by use of a novel electrophilic haloacetyl linker of formula R2xe2x80x94CH2xe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94(CH2)nxe2x80x94 wherein n is from 1 to about 10 and R2 is halogen, which is preferably chlorine.
In accordance with some preferred embodiments of the methods of the invention, a compound of Formula I: 
wherein the constituent variables are as previously defined, is reacted with the 5xe2x80x2-hydroxyl of a nucleoside, nucleotide, oligonucleotide, or oligonucleotide connected to a solid support as represented by Formula IV: 
to produce a phosphite compound of Formula IVa: 
In some preferred embodiments, the phosphite linkage is then oxidized or sulfurized by standard techniques to form a phosphotriester or phosphorothiotriester linkage, as represented by Formula IVb: 
wherein R11 is O or S.
The compound of Formula IVb is then contacted with a pendant group having, in preferred embodiments, a primary amino or thio group, for a time and under conditions sufficient to form the conjugate.
As used herein, the term xe2x80x9creactingxe2x80x9d means placing the indicated moieties together under conditions that will cause the moieties to perform the chemical reaction indicated.
As used herein, the term contacting means placing the indicated moieties together in a container under conditions that will cause the indicated reaction to occur.
As used herein, the term xe2x80x9cpendant groupxe2x80x9d means a functional or reporting group as is used in bioanalytical and/or antisense applications. Representative pendant groups include amines such as long and short straight, branched chain or cyclic alkylamines, polyamines such as spermine and spemidiine, polyalkylamines, thiols including aliphatic and aromatic mercaptans such as n-C18H37SH, thiocresol, benzylmercaptan, and thiocholesterol, proteins, peptides, and amino acids.
A wide variety of such pendant groups are amenable to the present invention. It is only required that the pendant group have a nucleophilic moiety such as an amino group, a sulfur atom, or an oxygen atom which is capable of displacing the halogen atom of the electrophilic linker and thus effecting the linkage of to the pendant group.
In some preferred embodiments, the haloacetyl linker is incorporated into an activated nucleoside phosphoramidite synthon (i.e., embodiments wherein variable xe2x80x9cvxe2x80x9d is 1 in the Formulas herein).
The electrophilic haloacetyl linker can be attached to an activated nucleoside phosphoramidite synthon at the sugar moiety thereof, preferably through the 2xe2x80x2-hydroxyl (e.g., embodiments wherein variable xe2x80x9cvxe2x80x9d is 1 and Q has Formula II). The electrophilic haloacetyl linker also can be attached to an activated nucleoside phosphoramidite synthon at the base moiety thereof, preferably through the N-2 nitrogen (e.g., embodiments wherein variable xe2x80x9cvxe2x80x9d is 1 and Q has Formula III).
The electrophilic haloacetyl linker also can be attached to the intersugar linkage of an oligonucleotide. Acordingly, in some preferred embodiments, the linker can be prepared as a phosphoramidite (e.g., embodiments wherein variable xe2x80x9cvxe2x80x9d is 0 in the Formulas herein) of Formula X: 
wherein the constituent variables are as described supra. Such compounds can be advantageously employed to attach the haloacetyl linker to, for example, the 5xe2x80x2-terminal hydroxyl of a completed oligonucleotide chain using standard phosphoramdite oligonucleotide synthetic regimes. Thus, in accordance with the methods of the invention, the haloacetyl linker can be conveniently introduced into an oligonucleotide during standard oligonucleotide synthesis at the 3xe2x80x2-terminus, the 5xe2x80x2-terminus, or at any position in the oligonucleotide.
The methods of the invention provide the additional feature that a pendant group of interest can be attached postsynthetically to the fully protected support-bound oligonucleotide via either thiol or primary amino group. In an additional advantage provided by the methods of the invention, the postsynthetic coupling of pendant group may be carried our both in aqueous and organic solvent.
In preferred embodiments, the concentration of the compound to be attached is from about 0.05M to about 1.0M.
It is preferred that the coupling of pendant groups through thiol groups be performed in the presence of a tertiary amine as a catalyst, preferably from about 0.5M to about 1.0M. Suitable preferred catalysts include triethylamine, ethyldiisopropylamine, and 1,8-diazabicyclo[4.5.0]undec-7-ene (DBU).
At the completion of the last coupling of the oligonucleotide synthetic cycle, the resulting oligonucleotide having the electrophilic haloacetyl linker attached thereto is typically coupled to a pendant group through the linker, and the completed oligomer is then released from the solid support and simultaneously deprotected by contacting with aqueous ammonia. Typical isolated yields of modified oligonucleotides is 20 to 70% depending on the length of the oligonucleotide, and the reactivity of particular pendant group.
The methods of the invention can be used to prepare oligomers having a variety of internucleoside linkages, represented by Z in the formulas herein. These include phosphodiester, phosphotriester, and phosphorothioate linkages.
The oligonucleotides prepared by the methods of the invention can have a wide variety of substitutents at their 2xe2x80x2-positions, in addition to the haloacetyl linker described herein. These include those described for substituent R6 of the formulas described herein.
The methods of the invention provide for oligonucleotides having one, two or a plurality of pendant groups attached through novel linkers described herein.
In some preferred embodiments, compounds of Formula X: 
are prepared by providing a compound of Formula H2Nxe2x80x94(CH2)nxe2x80x94OH wherein n is 1 to about 10;
reacting the compound with a compound of Formula VIII: 
to form a chloroacetylamino alkanol compound of Formula IX: 
and contacting said chloroacetylamino alkanol compound with a reagent of Formula (R3)2Pxe2x80x94Oxe2x80x94R1 for a time and under conditions sufficient to form the activated phosphate compound of Formula X: 
In preferred embodiments, the reaction of the compound of Formula H2Nxe2x80x94(CH2)nxe2x80x94OH with the compound of Formula VIII, and the contacting of the chloroacetylamino alkanol compound of Formula IX with the reagent of Formula (R3)2Pxe2x80x94Oxe2x80x94R1 is preferably performed in an organic solvent, the selection of which is within the skil of htose in the art.
The methods of the present invention can further include reacting the activated phosphate compound of formula X with a free hydroxyl group of a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support to form a compound of Formula XI: 
wherein R9 is a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support. Preferably, this is carried out in according to standard solid phase phosphoramidite synthetic protocols.
The phosphite compound of Formula XI is then preferably oxidizing or sulfurized to form a compound of Formula XII: 
wherein R11 is O or S, according to methodologies disclsoed supra. A pendant group is then coupled to the compound of Formula XII, as described supra.
The methods of the present invention are useful for the preparation of oligomeric compounds containing monomeric subunits that are joined by a variety of linkages, including phosphite, phosphodiester, phosphorothioate, and/or phosphorodithioate linkages. As used herein, the term xe2x80x9coligomeric compoundxe2x80x9d is used to refer to compounds containing a plurality of nucleoside monomer subunits that are joined by internucleoside linkages, preferably phosphorus-containing linkages, such as phosphite, phosphodiester, phosphorothioate, and/or phosphorodithioate linkages. The terms xe2x80x9coligomeric compoundxe2x80x9d or xe2x80x9coligomerxe2x80x9d therefore includes naturally occurring oligonucleotides, their analogs, and synthetic oligonucleotides. Monomer or higher order synthons having the Formulas described herein include both native (i.e., naturally occurring) and synthetic (e.g., modified native or totally synthetic) nucleosides and nucleotides.
Methods for coupling compounds of Formula I and Formula IV of the invention include both solution phase and, preferably, solid phase chemistries. Representative solution phase techniques are described in U.S. Pat. No. 5,210,264, which is assigned to the assignee of the present invention. In preferred embodiments, the methods of the present invention are employed for use in iterative solid phase oligonucleotide synthetic regimes. Representative solid phase techniques are those typically employed for DNA and RNA synthesis utilizing standard phosphoramidite chemistry, (see, e.g., Protocols For Oligonucleotides And Analogs, Agrawal, S., ed., Humana Press, Totowa, N.J., 1993, hereby incorporated by reference in its entirety). A preferred synthetic solid phase synthesis utilizes phosphoramidites as activated phosphate compounds. In this technique, a phosphoramidite monomer is reacted with a free hydroxyl on the growing oligomer chain to produce an intermediate phosphite compound, which is subsequently oxidized to the PV state using standard methods. This technique is commonly used for the synthesis of several types of linkages including phosphodiester, phosphorothioate, and phosphorodithioate linkages.
Typically, the first step in such a process is attachment of a first monomer or higher order subunit containing a protected 5xe2x80x2-hydroxyl to a solid support, usually through a linker, using standard methods and procedures known in the art. See for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991, hereby incorporated by reference in its entirety. The support-bound monomer or higher order first synthon is then treated to remove the 5xe2x80x2-protecting group, typically by treatment with acid. The solid support bound monomer is then reacted with a nucleoside phosphoramidite under anhydrous conditions in the presence of an activating agent such as, for example, 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, or diisopropylamino tetrazolide to form a phosphite triester linkage.
The phosphite triester linkage is subsequently oxidized or sulfurized. Choice of oxidizing or sulfurizing agent will determine whether the linkage will be oxidized or sulfurized to a phosphotriester, thiophosphotriester, or a dithiophosphotriester linkage.
It is generally preferable to perform a capping step, either prior to or after oxidation or sulfurization of the phosphite triester, thiophosphite triester, or dithiophosphite triester. Such a capping step is generally known to be beneficial by preventing shortened oligomer chains, by blocking chains that have not reacted in the coupling cycle. One representative reagent used for capping is acetic anhydride. Other suitable capping reagents and methodologies can be found in U.S. Pat. No. 4,816,571, issued Mar. 28, 1989, hereby incorporated by reference in its entirety.
Treatment with an acid removes the 5xe2x80x2-hydroxyl protecting group, and the synthetic cycle is repeated until the desired oligomer is achieved. At any point in the iterative cycle, a compound of the invention having, for example, Formula I, can be introduced into the oligonucleotide chain. Thus, the methods of the invention provide for the introduction of linked pendant groups at any position in the oligomeric chain.
After the coupling of the final phosphoramidite synthon, the completed oligomer is then cleaved from the solid support. The cleavage step, which can precede or follow deprotection of protected functional groups, will in preferred embodiments yield the completed oligomer free from the solid support, and devoid of all phosphorus protecting groups; that is, during cleavage, the linkages between monomeric subunits are converted from phosphotriester, thiophosphotriester, or dithiophosphotriester linkages to phosphodiester, phosphorothioate, or phosphorodithioate linkages.
The internucleoside linkages of the oligomeric compounds described herein, represented by moiety Z in the compounds and methods described herein, can be any internucleoside linkage as is known in the art, including phosphorus based linking groups such as phosphite, phosphodiester, phosphorothioate, and phosphorodithioate linkages. Such linkages can be protected, i.e., they can bear, for example, phosphorus protecting groups. As used herein, the term xe2x80x9cphosphorus protecting groupxe2x80x9d is intended to denote protecting groups that are known to be useful to protect phosphorus-containing linkages during oligonucleotide synthesis. One such preferred phosphorus protecting group is the xcex2-cyanoethyl protecting group.
Other representative phosphorus protecting groups include xe2x80x94CH2CHxe2x95x90CHCH2CN, para-CH2C6H4CH2CN, xe2x80x94(CH2)2-5N(H)COCF3, xe2x80x94CH2CH2Si(C6H5)2CH3, xe2x80x94CH2CH2N(CH3)COCF3 and others known in the art.
In preferred embodiments, the methods of the invention are used for the preparation of oligomeric compounds, preferably oligonucleotides. As used herein, the term xe2x80x9coligonuclotidexe2x80x9d means compounds that can contain both naturally occurring (i.e. xe2x80x9cnaturalxe2x80x9d) and non-naturally occurring (xe2x80x9csyntheticxe2x80x9d) moieties, for example, nucleosidic subunits containing modified sugar and/or nucleobase portions. Such oligonucleotide analogs are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides. Thus, oligonucleotide analogs include all such structures which function effectively to mimic the structure and/or function of a desired RNA or DNA strand, for example, by hybridizing to a target. The term synthetic nucleoside, for the purpose of the present invention, refers to a modified nucleoside. Representative modifications include modification of a heterocyclic base portion of a nucleoside to give a non-naturally occurring nucleobase, a sugar portion of a nucleoside, or both simultaneously.
Representative nucleobases useful in the compounds and methods described herein include adenine, guanine, cytosine, uridine, and thymine, as well as other non-naturally occurring and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine. Further naturally and non naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley and Sons, 1990, pages 858-859, Cook, P. D., Anti-Cancer Drug Design, 1991, 6, 585-607, each of which are hereby incorporated by reference in their entirety. The term xe2x80x98nucleosidic basexe2x80x99 is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain xe2x80x98universal basesxe2x80x99 that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as a universal base is 3-nitropyrrole.
Representative 2xe2x80x2 sugar modifications (position R1) amenable to the present invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula (O-alkyl)m, where m is 1 to about 10. Preferred among these polyethers are linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups, such as crown ethers and those which are disclosed by Ouchi, et al., Drug Design and Discovery 1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and Delgardo et. al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249, each of which are hereby incorporated by reference in their entirety. Further sugar modifications are disclosed in Cook, P. D., supra. Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino substitution is described in U.S. patent application Ser. No. 08/398,901, filed Mar. 6, 1995, entitled Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2xe2x80x2 and 5xe2x80x2 Substitutions, hereby incorporated by reference in its entirety.
Sugars having O-substitutions on the ribosyl ring are also amenable to the present invention. Representative substitutions for ring 0 include S, CH2, CHF, and CF2, see, e.g., Secrist, et al., Abstract 21, Program and Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications, Park City, Utah, Sep. 16-20, 1992, hereby incorporated by reference in its entirety.
As used herein, the term xe2x80x9calkylxe2x80x9d includes but is not limited to straight chain, branch chain, and alicyclic hydrocarbon groups. Alkyl groups of the present invention may be substituted. Representative alkyl substituents are disclosed in U.S. Pat. No. 5,212,295, at column 12, lines 41-50, hereby incorporated by reference in its entirety. As used herein, the term xe2x80x9clower alkylxe2x80x9d is intended to mean alkyl having 6 or fewer carbons.
As used herein, the term xe2x80x9caralkylxe2x80x9d denotes alkyl groups which bear aryl groups, for example, benzyl groups. The term xe2x80x9calkarylxe2x80x9d denotes aryl groups which bear alkyl groups, for example, methylphenyl groups. As used herein the term xe2x80x9carylxe2x80x9d denotes aromatic cyclic groups including but not limited to phenyl, naphthyl, anthracyl, phenanthryl, and pyrenyl.
As used herein, the term xe2x80x9calkanoylxe2x80x9d has its accustomed meaning as a group of formula xe2x80x94C(xe2x95x90O)-alkyl. A preferred alkanoyl group is the acetyl group.
In general, the term xe2x80x9cheteroxe2x80x9d denotes an atom other than carbon, preferably but not exclusively N, O, or S. Accordingly, the term xe2x80x9cheterocycloalkylxe2x80x9d denotes an alkyl ring system having one or more heteroatoms (i.e., non-carbon atoms). Preferred heterocycloalkyl groups include, for example, morpholino groups. As used herein, the term xe2x80x9cheterocycloalkenylxe2x80x9d denotes a ring system having one or more double bonds, and one or more heteroatoms. Preferred heterocycloalkenyl groups include, for example, pyrrolidino groups.
In some preferred embodiments of the invention R7 can be a linker connected to a solid support. Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069. Linkers are known in the art as short molecules which serve to connect a solid support to functional groups (e.g., hydroxyl groups) of initial synthon molecules in solid phase synthetic techniques. Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991, Chapter 1, pages 1-23.
Solid supports according to the invention include those generally known in the art to be suitable for use in solid phase methodologies, including, for example, controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527, hereby incorporated by reference in its entirety), TentaGel Supportxe2x80x94an aminopolyethyleneglycol derivatized support (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373, hereby incorporated by reference in its entirety) and Porosxe2x80x94a copolymer of polystyrene/divinylbenzene.
In some preferred embodiments of the invention R7 or R5 can be a hydroxyl protecting group. A wide variety of hydroxyl protecting groups can be employed in the methods of the invention. Preferably, the protecting group is stable under basic conditions but can be removed under acidic conditions. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting groups are disclosed by Beaucage, et al., Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley and Sons, New York, 1991, each of which are hereby incorporated by reference in their entirety. Preferred protecting groups used for R7 or R5 include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). The R7 or R5 group can be removed from oligomeric compounds of the invention by techniques well known in the art to form the free hydroxyl.
For example, dimethoxytrityl protecting groups can be removed by protic acids such as formic acid, dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid or with Lewis acids such as for example zinc bromide. See for example, Greene and Wuts, supra.
In some preferred embodiments of the invention amino groups are appended to alkyl or to other groups such as, for example, to 2xe2x80x2-alkoxy groups. Such amino groups are also commonly present in naturally occurring and non-naturally occurring nucleobases. It is generally preferred that these amino groups be in protected form during the synthesis of oligomeric compounds of the invention. epresentative amino protecting groups suitable for these purposes are discussed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 7, 2d ed, John Wiley and Sons, New York, 1991. Generally, as used herein, the term xe2x80x9cprotectedxe2x80x9d when used in connection with a molecular moiety such as xe2x80x9cnucleobasexe2x80x9d indicates that the molecular moiety contains one or more functionalities protected by protecting groups.
Sulfurizing agents used during oxidation to form phosphorothioate and phosphorodithioate linkages include Beaucage reagent (see e.g. Iyer, R. P., et. al., J. Chem. Soc., 1990, 112, 1253-1254, and Iyer, R. P., et. al., J. Org. Chem., 1990, 55, 4693-4699); tetraethylthiuram disulfide (see e.g., Vu, H., Hirschbein, B. L., Tetrahedron Lett., 1991, 32, 3005-3008); dibenzoyl tetrasulfide (see e.g., Rao, M. V., et. al., Tetrahedron Lett., 1992, 33, 4839-4842); di(phenylacetyl)disulfide (see e.g., Kamer, P. C. J., Tetrahedron Lett., 1989, 30, 6757-6760); Bis(O,O-diisopropoxy phosphinothioyl)disulfids (see Stec et al., Tetrahedron Lett., 1993, 34, 5317-5320); 3-ethoxy-1,2,4-dithiazoline-5-one (see Nucleic Acids Research, 1996 24, 1602-1607, and Nucleic Acids Research, 1996 24, 3643-3644); Bis(p-chlorobenzenesulfonyl)disulfide (see Nucleic Acids Research, 1995 23, 4029-4033); sulfur, sulfur in combination with ligands like triaryl, trialkyl, triaralkyl, or trialkaryl phosphines. The foregoing references are hereby incorporated by reference in their entirety.
Useful oxidizing agents used to form the phosphodiester or phosphorothioate linkages include iodine/tetrahydrofuran/water/pyridine or hydrogen peroxide/water or tert-butyl hydroperoxide or any peracid like m-chloroperbenzoic acid. In the case of sulfurization the reaction is performed under anhydrous conditions with the exclusion of air, in particular oxygen whereas in the case of oxidation the reaction can be performed under aqueous conditions.
Oligonucleotides or oligonucleotide analogs according to the present invention are preferably hybridizable to a specific target preferably comprise from about 5 to about 50 monomer subunits. It is more preferred that such compounds comprise from about 10 to about 30 monomer subunits, with 15 to 25 monomer subunits being particularly preferrred. When used as xe2x80x9cbuilding blocksxe2x80x9d in assembling larger oligomeric compounds (i.e., as synthons of Formula II), smaller oligomeric compounds are preferred. Libraries of dimeric, trimeric, or higher order compounds of general Formula II can be prepared for use as synthons in the methods of the invention. The use of small sequences synthesized via solution phase chemistries in automated synthesis of larger oligonucleotides enhances the coupling efficiency and the purity of the final oligonucloetides. See for example: Miura, K., et al., Chem. Pharm. Bull., 1987, 35, 833-836; Kumar, G., and Poonian, M. S., J. Org. Chem., 1984, 49, 4905-4912; Bannwarth, W., Helvetica Chimica Acta, 1985, 68, 1907-1913; Wolter, A., et al., nucleosides and nucleotides, 1986, 5, 65-77, each of which are hereby incorporated by reference in their entirety.
In one aspect of the invention, the compounds of the invention are used to modulate RNA or DNA, which code for a protein whose formation or activity it is desired to modulate. The targeting portion of the composition to be employed is, thus, selected to be complementary to the preselected portion of DNA or RNA, that is to be hybridizable to that portion.
Compounds having chiral phosphorus linkages are contemplated by the present invention. See Stec, W. J., and Lesnikowski, Z. J., in Methods in Molecular Biology Vol. 20: Protocols for Oligonucleotides and Analogs, S. Agrawal, Ed., Humana Press, Totowa, N.J. (1993), at Chapter 14. See also Stec, W. J. et al., Nucleic Acids Research, Vol. 19, No. 21, 5883-5888 (1991); and European Patent Application EP 0 506 242 Al, each of which are hereby incorporated by reference in their entirety.
The oligomeric compounds of the invention can be used in diagnostics, therapeutics and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms, including warm-blooded animals, can be treated. Further, each cell of multicellular eukaryotes can be treated, as they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Furthermore, many of the organelles (e.g., mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms. Thus, single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides.
In preferred embodiments, compounds of the invention comprising the haloacetyl linker include those having the Formula V: 
wherein:
R2 is halogen which is preferably chlorine, or R2 is a pendant group;
R10 is a nucleobase, a nucleoside, a nucleotide, an activated nucleotide, an oligonucleotide, an oligonucleotide connected to a solid support, or a moiety of Formula VI: 
wherein:
R1 is H or a phosphorus protecting group;
R3 is xe2x80x94N(R4)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R4 is straight or branched chain alkyl having from 1 to 10 carbons; and
n is from 1 to about 10.
In more preferred embodiments, compounds of the invention having the Formula VII: 
wherein:
v and q are each 0 or 1, provided that the sum of v and q is not 0;
q is 0 or 1;
Q has one of the Formulas II or III: 
wherein:
R5 is a hydroxyl protecting group;
B is a nucleobase;
R6 is F, Oxe2x80x94R20, Sxe2x80x94R20 or Nxe2x80x94R20(R21);
R20 is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic;
and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylamino, urea or alkylurea group;
or R20 has one of the formulas: 
wherein
Q1 is O, S or NR2;
m is from 1 to 10;
y is from 0 to 10;
E is N (R21) (R31), Nxe2x95x90C(R21)(R31), C1-C10 alkyl, or C1-C10 substituted alkyl wherein said substituent is N (R2) (R3);
each R21 and R31 is, independently, H, C1-C10 alkyl, alkylthioalkyl, a nitrogen protecting group, or R2 and R3, together, are a nitrogen protecting group or wherein R2 and R3 are joined in a ring structure that can include at least one heteroatom selected from N and O; and R41 is H or C1-C12 alkyl.
As will be recognized, the steps of the methods of the present invention need not be performed any particular number of times or in any particular sequence. Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are intended to be illustrative and not intended to be limiting.